Skip to main content

Advertisement

SpringerLink
Log in

Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations

  • Original Article
  • Published:
Journal of Endocrinological Investigation Aims and scope Submit manuscript

Abstract

Purpose

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease characterized by impaired adrenal steroidogenesis and most often caused by CYP21A2 gene mutations. For the first time, we reported complete spectrum and frequency of CYP21A2 gene mutations in 61 unrelated patients with classical and non-classical CAH from Serbia.

Methods

Direct DNA sequencing of whole CYP21A2 gene and polymerase chain reaction with sequence-specific primers for detection of CYP21A1P/CYP21A2 chimeras were combined.

Results

We identified 18 different pathogenic alleles—two of them novel. Mutation detection rate was highest in patients with salt-wasting form of CAH (94.7 %). The most prevalent mutation was intron 2 splice site mutation, c.290-13A/C>G (18.5 %). Other mutation frequencies were: CYP21A1P/CYP21A2 chimeras (13 %), p.P30L (13 %), p.R356W (11.1 %), p.G110fs (7.4 %), p.Q318X (4.6 %), p.V281L (4.6 %), p.I172N (2.8 %), p.L307fs (2.8 %), p.P453S (1.9 %), etc. Mainly, frequencies were similar to those in Slavic populations and bordering countries. However, we found 6.5 % of alleles with multiple mutations, frequently including p.P453S. Effects of novel mutations, c.386T>C (p.Leu129Pro) and c.493T>C (p.Ser165Pro), were characterized in silico as deleterious. The effect of well-known mutations on Serbian patients’ phenotype was as expected.

Conclusions

The first comprehensive molecular genetic study of Serbian CAH patients revealed two novel CYP21A2 mutations. This study will enable genetic counseling in our population and contribute to better understanding of molecular landscape of CAH in Europe.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

Abbreviations

17-OHP:

17-Hydroprogesterone

21-OH:

21-Hydroxylase

Bp:

Base pair

CAH:

Congenital adrenal hyperplasia

CYP21A2:

Cytochrome P450, family 21, subfamily A, polypeptide 2

dNTP:

Deoxyribonucleoside triphosphate

MLPA:

Multiplex ligation-dependent probe amplification

HGMD:

Human gene mutation database

NC-CAH:

Non-classical form of CAH

PCR:

Polymerase chain reaction

PCR-SSP:

Sequence-specific PCR amplification

SV-CAH:

Simple virilizing form of CAH

SW-CAH:

Salt-wasting form of CAH

UTR:

Untranslated region

References

  1. Concolino P, Mello E, Zuppi C, Capoluongo E (2010) Molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: an update of new CYP21A2 mutations. Clin Chem Lab Med 48:1057–1062. doi:10.1515/cclm.2010.239

    Article  CAS  PubMed  Google Scholar 

  2. Gonçalves J, Friães A, Moura L (2007) Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency. Expert Rev Mol Med 9:1–23. doi:10.1017/s1462399407000300

    Article  PubMed  Google Scholar 

  3. Rabbani B, Mahdieh N, Haghi Ashtiani MT, Akbari MT, Rabbani A (2011) Molecular diagnosis of congenital adrenal hyperplasia in Iran: focusing on CYP21A2 gene. Iran J Pediatr 21:139–150

    PubMed Central  PubMed  Google Scholar 

  4. Trapp CM, Oberfield SE (2012) Recommendations for treatment of nonclassic congenital adrenal hyperplasia (NCCAH): an update. Steroids 77:342–346. doi:10.1016/j.steroids.2011.12.009

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Yang Z, Mendoza AR, Welch TR, Zipf WB, Yu CY (1999) Modular variations of the human major histocompatibility complex class III genes for serine/threonine kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and tenascin TNX (the RCCX module). A mechanism for gene deletions and disease associations. J Biol Chem 274:12147–12156

    Article  CAS  PubMed  Google Scholar 

  6. New MI, Lekarev O, Mancenido D, Parsa A, Yuen T (2013) Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. In: New MI, Lekarev O, Parsa A, O’Malley B, Hammer GD (eds) Genetic steroid disorders. Elsevier, New York, pp 29–51

    Google Scholar 

  7. Tusié-Luna MT, White PC (1995) Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms. Proc Natl Acad Sci USA 92:10796–10800

    Article  PubMed Central  PubMed  Google Scholar 

  8. Krone N, Arlt W (2009) Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 23:181–192. doi:10.1016/j.beem.2008.10.014

    Article  CAS  PubMed  Google Scholar 

  9. Sharaf S, Hafez M, ElAbd D, Ismail A, Thabet G, Elsharkawy M (2014) High frequency of splice site mutation in 21-hydroxylase deficiency children. J Endocrinol Invest. doi:10.1007/s40618-014-0207-1

    PubMed  Google Scholar 

  10. Krone N, Braun A, Weinert S, Peter M, Roscher AA, Partsch CJ, Sippell WG (2002) Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia. Clin Chem 48:818–825

    CAS  PubMed  Google Scholar 

  11. Dolzan V, Stopar-Obreza M, Zerjav-Tansek M, Breskvar K, Krzisnik C, Battelino T (2003) Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease. Eur J Endocrinol 149:137–144

    Article  CAS  PubMed  Google Scholar 

  12. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi:10.1038/msb.2011.75

    Article  PubMed Central  PubMed  Google Scholar 

  13. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi:10.1093/bioinformatics/bti770

    Article  CAS  PubMed  Google Scholar 

  14. Robins T, Carlsson J, Sunnerhagen M, Wedell A, Persson B (2006) Molecular model of human CYP21 based on mammalian CYP2C5: structural features correlate with clinical severity of mutations causing congenital adrenal hyperplasia. Mol Endocrinol 20:2946–2964. doi:10.1210/me.2006-0172

    Article  CAS  PubMed  Google Scholar 

  15. Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna MT, Lesser M, New MI, White PC (1992) Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 90:584–595. doi:10.1172/jci115897

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Wedell A, Thilén A, Ritzén EM, Stengler B, Luthman H (1994) Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 78:1145–1152. doi:10.1210/jcem.78.5.8175971

    CAS  PubMed  Google Scholar 

  17. Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP (2000) Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 85:1059–1065. doi:10.1210/jcem.85.3.6441

    Article  CAS  PubMed  Google Scholar 

  18. Cavarzere P, Vincenzi M, Teofoli F, Gaudino R, Lauriola S, Maines E, Camilot M, Antoniazzi F (2013) Genotype in the diagnosis of 21-hydroxylase deficiency: who should undergo CYP21A2 analysis? J Endocrinol Invest 36:1083–1089. doi:10.3275/9096

    CAS  PubMed  Google Scholar 

  19. Balraj P, Lim PG, Sidek H, Wu LL, Khoo AS (2013) Mutational characterization of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Malaysia. J Endocrinol Invest 36:366–374. doi:10.3275/8648

    CAS  PubMed  Google Scholar 

  20. Wilson RC, Mercado AB, Cheng KC, New MI (1995) Steroid 21-hydroxylase deficiency: genotype may not predict phenotype. J Clin Endocrinol Metab 80:2322–2329. doi:10.1210/jcem.80.8.7629224

    CAS  PubMed  Google Scholar 

  21. Pérez B, Rodríguez-Pascau L, Vilageliu L, Grinberg D, Ugarte M, Desviat LR (2010) Present and future of antisense therapy for splicing modulation in inherited metabolic disease. J Inherit Metab Dis 33:397–403. doi:10.1007/s10545-010-9135-1

    Article  PubMed  Google Scholar 

  22. Wilson RC, Nimkarn S, Dumic M, Obeid J, Azar MR, Azar M, Najmabadi H, Saffari F, New MI (2007) Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Mol Genet Metab 90:414–421. doi:10.1016/j.ymgme.2006.12.005

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Anastasovska V, Kocova M (2010) Genotype-phenotype correlation in CAH patients with severe CYP21A2 point mutations in the Republic of Macedonia. J Pediatr Endocrinol Metab 23:921–926

    Article  CAS  PubMed  Google Scholar 

  24. Dolzan V, Sólyom J, Fekete G, Kovács J, Rakosnikova V, Votava F, Lebl J, Pribilincova Z, Baumgartner-Parzer SM, Riedl S, Waldhauser F, Frisch H, Stopar-Obreza M, Krzisnik C, Battelino T (2005) Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia. Eur J Endocrinol 153:99–106. doi:10.1530/eje.1.01944

    Article  CAS  PubMed  Google Scholar 

  25. Grigorescu Sido A, Weber MM, Grigorescu Sido P, Clausmeyer S, Heinrich U, Schulze E (2005) 21-Hydroxylase and 11beta-hydroxylase mutations in Romanian patients with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab 90:5769–5773. doi:10.1210/jc.2005-0379

    Article  PubMed  Google Scholar 

  26. Baş F, Kayserili H, Darendeliler F, Uyguner O, Günöz H, Yüksel Apak M, Atalar F, Bundak R, Wilson RC, New MI, Wollnik B, Saka N (2009) CYP21A2 gene mutations in congenital adrenal hyperplasia: genotype-phenotype correlation in Turkish children. J Clin Res Pediatr Endocrinol 1:116–128. doi:10.4008/jcrpe.v1i3.49

    PubMed Central  PubMed  Google Scholar 

  27. Dracopoulou-Vabouli M, Maniati-Christidi M, Dacou-Voutetakis C (2001) The spectrum of molecular defects of the CYP21 gene in the Hellenic population: variable concordance between genotype and phenotype in the different forms of congenital adrenal hyperplasia. J Clin Endocrinol Metab 86:2845–2848. doi:10.1210/jcem.86.6.7574

    CAS  PubMed  Google Scholar 

  28. Barbat B, Bogyo A, Raux-Demay MC, Kuttenn F, Boué J, Simon-Bouy B, Serre JL, Mornet E (1995) Screening of CYP21 gene mutations in 129 French patients affected by steroid 21-hydroxylase deficiency. Hum Mutat 5:126–130. doi:10.1002/humu.1380050205

    Article  CAS  PubMed  Google Scholar 

  29. Carrera P, Bordone L, Azzani T, Brunelli V, Garancini MP, Chiumello G, Ferrari M (1996) Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency. Hum Genet 98:662–665

    Article  CAS  PubMed  Google Scholar 

  30. Ezquieta B, Oliver A, Gracia R, Gancedo PG (1995) Analysis of steroid 21-hydroxylase gene mutations in the Spanish population. Hum Genet 96:198–204

    Article  CAS  PubMed  Google Scholar 

  31. Finkielstain GP, Chen W, Mehta SP, Fujimura FK, Hanna RM, Van Ryzin C, McDonnell NB, Merke DP (2011) Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 96:E161–E172. doi:10.1210/jc.2010-0319

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Dain LB, Buzzalino ND, Oneto A, Belli S, Stivel M, Pasqualini T, Minutolo C, Charreau EH, Alba LG (2002) Classical and nonclassical 21-hydroxylase deficiency: a molecular study of Argentine patients. Clin Endocrinol (Oxf) 56:239–245

    Article  CAS  Google Scholar 

  33. Haider S, Islam B, D’Atri V, Sgobba M, Poojari C, Sun L, Yuen T, Zaidi M, New MI (2013) Structure-phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia. Proc Natl Acad Sci USA 110:2605–2610. doi:10.1073/pnas.1221133110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, Chitayat D, Sun L, Zaidi M, Wilson RC, Yuen T (2013) Genotype-phenotype correlation in 1507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci USA 110:2611–2616. doi:10.1073/pnas.1300057110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. White PC, Speiser PW (2000) Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 21:245–291. doi:10.1210/edrv.21.3.0398

    CAS  PubMed  Google Scholar 

  36. Kleinle S, Lang R, Fischer GF, Vierhapper H, Waldhauser F, Födinger M, Baumgartner-Parzer SM (2009) Duplications of the functional CYP21A2 gene are primarily restricted to Q318X alleles: evidence for a founder effect. J Clin Endocrinol Metab 94:3954–3958. doi:10.1210/jc.2009-0487

    Article  CAS  PubMed  Google Scholar 

  37. Lekarev O, Tafuri K, Lane AH, Zhu G, Nakamoto JM, Buller-Burckle AM, Wilson TA, New MI (2013) Erroneous prenatal diagnosis of congenital adrenal hyperplasia owing to a duplication of the CYP21A2 gene. J Perinatol 33:76–78. doi:10.1038/jp.2012.5

    Article  CAS  PubMed  Google Scholar 

  38. Dipple KM, McCabe ER (2000) Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modifiers, and systems dynamics. Am J Hum Genet 66:1729–1735. doi:10.1086/302938

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Scriver CR, Waters PJ (1999) Monogenic traits are not simple: lessons from phenylketonuria. Trends Genet 15:267–272

    Article  CAS  PubMed  Google Scholar 

  40. Chen W, Xu Z, Sullivan A, Finkielstain GP, Van Ryzin C, Merke DP, McDonnell NB (2012) Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency. Clin Chem 58:421–430. doi:10.1373/clinchem.2011.174037

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, 11010, Serbia

    I. Milacic, M. Ugrin, K. Klaassen, A. Skakic, S. Pavlovic & M. Stojiljkovic

  2. Clinic of Endocrinology, Clinical Center of Serbia, School of Medicine, University of Belgrade, Doktora Subotića 13, Belgrade, 11000, Serbia

    M. Barac & S. Vujovic

  3. Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Radoja Dakića 6-8, Belgrade, 11070, Serbia

    T. Milenkovic, K. Mitrovic & S. Todorovic

  4. University Children’s Hospital, Tirsova 10, Belgrade, 11000, Serbia

    M. Jesic

  5. University Clinic for Obstetrics and Gynecology “Narodni Front”, Kraljice Natalije 62, Belgrade, 11000, Serbia

    I. Joksic

Authors
  1. I. Milacic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Barac
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. Milenkovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Ugrin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K. Klaassen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Skakic
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Jesic
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. I. Joksic
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. K. Mitrovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. S. Todorovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. S. Vujovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. S. Pavlovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. M. Stojiljkovic
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Stojiljkovic.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This study was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III41004).

Ethical approval

This study involved human participants and has been approved by the Ethics Committee of the School of Medicine, University of Belgrade in Belgrade, Serbia and the Ethics Committee of the Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic” in Belgrade, Serbia, and has therefore, been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Milacic, I., Barac, M., Milenkovic, T. et al. Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations. J Endocrinol Invest 38, 1199–1210 (2015). https://doi.org/10.1007/s40618-015-0366-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40618-015-0366-8

Keywords

Search

Navigation